Natamycin dosage form, method for preparing same and use thereof

ABSTRACT

The present invention relates to a novel natamycin dosage form for the food industry, and more particularly to microcapsules where natamycin is encapsulated within a physiologically acceptable shell to provide a protected natamycin product. The present invention relates also to novel processes for preparing the capsules according to the invention, as well as to the use of the capsules of the present invention. The invention further relates to food products containing natamycin in encapsulated form.

CLAIM OF PRIORITY

This application claims priority under 35 U.S.C. § 119 to GB applicationno. 0319817.3, filed Aug. 22, 2003, the entire contents of which havebeen incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to natamycin dosage forms for the foodindustry, and more particularly to microcapsules where natamycin as theactive food preservative ingredient is encapsulated within a shell. Thepresent invention relates also to novel methods for preparing themicrocapsules according to the invention, to the use of themicrocapsules of the present invention in the food industry, as well asto food products containing the same. Preferred food products includeacidic food products and sliced bread.

BACKGROUND OF THE INVENTION

Natamycin is a polyene macrolide natural antifungal agent produced byfermentation of the bacterium Streptomyces natalensis. Natamycin(previously known as pimaricin) has an extremely effective and selectivemode of action against a very broad spectrum of common food spoilageyeasts and moulds with most strains being inhibited by concentrations of1-15 ppm of natamycin.

Natamycin is accepted as a food preservative and used world wide,particularly for surface treatment of cheese and dried fermentedsausages. It has several advantages as a food preservative, includingbroad activity spectrum, efficacy at low concentrations, lack ofresistance, and activity over a wide pH range. Neutral aqueoussuspensions of natamycin are quite stable, but natamycin has poorstability in acid or alkaline conditions, in the presence of light,oxidants and heavy metals. For example, natamycin can be used inpasteurised fruit juice to prevent spoilage by heat-resistant mouldssuch as Byssochlamys. The acid pH of the juice, however, promotesdegradation of natamycin during pasteurisation as well as during storageif the juice is not refrigerated. Natamycin is also degraded by hightemperature heat processing, such as occurs during cooking of bakeryitems in an oven.

At extreme pH conditions, such as pH less than 4 and greater than 10,natamycin is rapidly inactivated with formation of various kinds ofdecomposition products. Acid hydrolysis of natamycin liberates theinactive aminosugar mycosamine. Further degradation reactions result information of dimers with a triene rather than a tetraene group. Heatingat low pH may also result in decarboxylation of the aglycone. Alkalinehydrolysis results in saponification of the lactone. Both aciddegradation products (aponatamycin, the aglycone dimer, and mycosamine),and alkaline or UV degradation products proved even safer than natamycinin toxicology tests, but are inactive biologically.

Natamycin is generally dosed into or onto food as a powder or as anaqueous natamycin suspension. This kind of dosage form leaves thenatamycin unprotected under the conditions of processing and use. Thenatamycin powder, although mixed with excipients such as lactose, mayalso be sticky to handle and cause dust problems within the foodprocessing plants. Furthermore, natamycin is so highly effective as anantifungal compound that it may adversely affect the processing of theproducts that it is intended to preserve if this is dependent on desiredfungal activity. There is thus a need for a protected dosage form ofnatamycin.

A general description of natamycin and its current uses may be found inThomas, L. V. and Delves-Broughton, J. 2003. Natamycin. In: Encyclopediaof Food Sciences and Nutrition. Eds. B. Caballero, L. Trugo and P.Finglas, pp 4109-4115. Elsevier Science Ltd.

Encapsulation technology has been applied to a number of foodingredients, usually to mask flavour or activity. The present inventionis based on the realization that unexpected benefits are derivable fromthe encapsulation of natamycin.

Koontz & Marcey, 2003, J Agric Food Chemistry 51: 7106-7110 describesthe formation of a natamycin/cyclodextrin inclusion product. Thecyclodextrin acts as host molecules to protect mainly against light, butalso low pH, heat and oxidation. However, this natamycin/cyclodextrincomplex is not a true encapsulation. A molecule of natamycin will not‘fit’ into the cavity of gamma-cyclodextrin, thus it can only beconsidered a partial encapsulation. Acidic conditions tend todestabilise this kind of complex, releasing the contents of thecyclodextrin molecule and the natamycin molecule is not completelyenclosed and protected by the cyclodextrin molecules. Koontz et al.2003. J Agric Food Chemistry 51: 7111-7114 has also described thestability of natamycin and its cyclodextrin inclusion complexes inaqueous solution.

EP115618 describes an anti-caking antimycotic food ingredient whereinthe anti-caking agent is encapsulated and then treated with natamycin toprovide antimycotic activity.

U.S. Pat. No. 5,445,949 describes a process for recovery of natamycin byseparation of a hydrophobic fermentation product such as natamycin. Theprocess involves a step including encapsulation of a protein but thereis no mention of encapsulating the natamycin.

EP-A1-1382261 describes the use of microbial inhibitors such asnatamycin for baked bread products, including shelf stable kits formaking snacks or meals. The microbial inhibitor is not protected byencapsulation.

Copending patent application U.S. Ser. No. 10/765,210, filed Jan. 28,2004 relates to the protection of fine bakery goods by spraying thesurface of the goods with a natamycin suspension and thus to increasethe shelf life of the products.

WO 89/033208 describes a polyene macrolide powder for liposomepreparation. The polyene macrolide is encapsulated in liposomes in orderto modify the pharmokinetics of the antifungal in systemic diseases. Theliposome is intended for pharmaceutical use only.

U.S. Pat. No. 5,821,233 concerns an antifungal composition whereinnatamycin is incorporated in porous silica to provide delayed release ofnatamycin in an aqueous medium.

General descriptions of encapsulation processes may be found in Shahidi,F., and X.-Q., Han. 1993. Encapsulation of food ingredient. CriticalReviews in Food Science and Nutrition 33 (6): 501-547.

The encapsulation of mold inhibitors is described by Ranum, P., 1999.Encapsulated mold inhibitors—the greatest thing since sliced bread inCereal Foods World, Vol 44, No 5, p. 370-371.

U.S. Pat. No. 5,204,029 discloses a process for preparing ediblemicrocapsules which contain a multiplicity of liquid cores. In theprocess, a water-in-oil emulsion, with the active ingredient dissolvedin an inner aqueous phase, is spray cooled, which causes thesolidification of the fat phase and the entrapment of the aqueous phaseas minute droplets dispersed in a microcapsule. This process, however,leads to very unstable microcapsules from which the water phase migratesfrom the inner part of the microcapsule to an outer part. This furtherresults in the condensation of the water on the wall of a container.

Kirk-Othmer Encyclopedia of Chemical Technology, 3^(rd) ed. Vol. 15, pp.473 to 474, discloses a process in which liquids are encapsulated usinga rotating extrusion head containing concentric nozzles. The process isonly suitable for liquids or slurries, and the products of the processare large beads having meltable coatings, such as fats or waxes.However, the microcapsules containing a single liquid droplet as a coreare very susceptible to rupture.

In their article “Mass preparation and characterization of alginatemicrospheres” in Process Biochemistry 35 (2000) 885 to 888 Mofidi, N. etal. describe a method for mass preparation of microspheres, in whichmethod a sterilized alginate solution is prepared and the solution isthen poured into a reactor containing a non-aqueous phase, while beingstirred. An emulsion of alginate microdroplets is formed and anappropriate amount of the cross-linker is added. Microsphericalginate-gel particles fell to the bottom and they were collected byfiltration.

Similarly, Wong, T. W. et al in J. Microencapsulation, 2002 Vol. 19, no4, 511 to 522, describe release characteristics from pectinmicrosphperes and the method for preparing these microspheres. In thismethod, pectin microspheres are prepared by a water-in-oil emulsiontechnique, in which minute droplets of pectin containing an activeingredient dispersed in a liquid hydrophobic continuous phase arehardened and collected by filtration.

Microencapsulation by a coacervation-phase separation process is knownfrom an article by Joseph A. Bakan in Controlled Release Technologies,1980 by Agis F. Kydonieus. The process consists of a series of threesteps carried out under continuous agitation: (1) formation of threeimmiscible chemical phases; (2) deposition of the coating; and (3)rigidization of the coating.

Sanghvi, S. P. and Nairn J. G. have studied the effect of viscosity andinterfacial tension on the particle size of cellulose acetatetrimellitate microspheres. The results are presented in their article inJ. Microencapsulation, 1992, Vol. 9, no 2, 215 to 227.

In their article in Lebensm. -Wiss. u. -Technol., 33, 80 to 88 (2000)Lee, S. J. and Rosenberg, M. describe a double emulsification and heatgelation process for preparing whey protein-based microcapsules. Themicrocapsules prepared according to the described process are wheyprotein-based microcapsules containing an apolar core material.

In their article in Science Vol. 298, 1 Nov. 2002, Dinsmore et al.describe selectively permeable capsules composed of colloidal particles.The capsules are fabricated by the self-assembly of colloidal particlesonto the interface of emulsion droplets. After the particles are lockedtogether to form elastic shells, the emulsion droplets are transferredto a fresh continuous-phase fluid that is the same as that inside thedroplets.

The documents mentioned in this specification should be consideredincorporated herein by reference.

A problem associated with the prior art natamycin dosage forms is thatthey leave the natamycin unprotected. The efficacy and application ofnatamycin is compromised by the lability of the preservative toconditions of heat, high and low pH, light and oxidation. There is aneed to protect the natamycin and also to provide release of the activenatamycin in a controlled manner from the dosage form.

The present invention seeks to overcome the problems of the knownnatamycin dosage forms, as described above, by providing capsules whichare stable against processing conditions and which provide a controlledand/or sustained release of the natamycin.

BRIEF DESCRIPTIONS OF THE INVENTION

The present invention is based on the use of encapsulation to protectthe natamycin molecule from degradation during adverse conditions and/orto protect process ingredients from exposure to natamycin duringprocessing. The present invention comprises the encapsulation ofnatamycin by various processes in order to protect it from suchdegradation or in order to protect the ingredients from such exposure,and the encapsulated natamycin food product itself and its use as a foodpreservative.

An object of the present invention is thus to provide a natamycin dosageform comprising microcapsules, where natamycin is encapsulated within aphysiologically acceptable shell to provide a protected foodpreservative natamycin product.

An object of the invention is also a process for producing a natamycindosage form comprising co-processing natamycin with a physiologicallyacceptable encapsulating material to cause said material to encapsulatesaid natamycin within a shell, and recovering a protected foodpreservative natamycin product.

A further object of the invention is a method for the preservation of afood product comprising adding to the food product an effectivefood-preserving amount of natamycin which is encapsulated within aphysiologically acceptable shell.

A further object of the invention is a preserved food product whichcomprises as a preservative an effective food preserving amount ofnatamycin which is encapsulated within a shell. The food product ispreferably selected from salad dressings, acidic dairy products(including natural cheese, cottage cheese, acidified cheese, creamcheese, yoghurt, sour cream, processed cheese), fruit juices, acidicdrinks, alcoholic drinks (including wine and beer), chilled dough andcooked or uncooked bakery products, dairy fillings and toppings forbaked goods, surface glazes and coatings for bakery items and otherheat-processed items, condiments, dips, purees, pickles, marinades,marinated meat or poultry, breaded meat or poultry, pizza toppings andbases, fast food products, kits for making snacks or meals, kits formaking bakery products, pet food, broiler feed and any other acidic,heat-processed and/or fungal fermented food products.

An especially preferred preserved food product is a sliced or cut bakeryproduct, especially sliced bread, wherein encapsulated natamycin hasbeen incorporated into the dough before cooking and providespreservation of the bakery product after baking.

Another preferred preserved food product comprises an acidic foodproduct, into which pH-protected natamycin of the present invention hasbeen incorporated.

The objects of the invention are achieved by the microcapsules,processes, methods and products defined in the independent claims.Preferred embodiments of the invention are disclosed in the dependentclaims.

The invention is based on the concept of protecting the active natamyciningredient by encapsulating it within a physiologically acceptable shellmaterial, for example a hydrophobic material or a hydrocolloid or anyother suitable encapsulating material or a mixture or combinationthereof.

The encapsulation of the natamycin is performed by processes which arenovel in combination with natamycin and which provide unexpectedbenefits to the food industry. The encapsulation processes andencapsulating materials or shell materials are selected depending on thenature of the continuous phase in the food application. The shellmaterial must be water-insoluble if the continuous phase of the foodapplication is water-based, and vice-versa in order to provide slowand/or delayed release as well as protection/segregation.

Suitable encapsulating processes comprise fluidized bed processes,liposome encapsulation processes, spray drying processes, spray coolingprocesses, extrusion processes, co-extrusion processes (such ascentrifugal co-extrusion), coacervation processes and combinationsthereof.

In a special double encapsulation process, the present inventionprovides a microcapsule which comprises a solidified hydrophobic shellmatrix, an encapsulated aqueous bead or beads encapsulated in thesolidified hydrophobic shell matrix, and natamycin as an activeingredient incorporated in the encapsulated aqueous bead or beads.

This natamycin dosage form is provided by a double encapsulation methodfor preparing microcapsules, which method comprises the steps of

-   a) providing an aqueous phase and natamycin incorporated in the    aqueous phase,-   b) providing a hydrophobic phase in melted form,-   c) incorporating or dissolving an encapsulating material or mixture    of encapsulating materials in the aqueous phase or in the    hydrophobic phase,-   d) combining the aqueous phase with the hydrophobic phase and    homogenizing or mixing the combined phases to form a water-in-oil    emulsion,-   e) encapsulating the aqueous phase in the emulsion, thus converting    the liquid aqueous phase into encapsulated aqueous beads, whereby a    dispersion comprising aqueous beads is formed and the natamycin is    incorporated in the aqueous beads, and-   f) processing the dispersion obtained in step e) to form    microcapsules where the encapsulated aqueous beads are further    encapsulated in the solidified hydrophobic shell matrix.

The encapsulation process of the present invention may also includegelling, cross-linking, coacervation, sintering or any other suitablemeans. In the above double encapsulation this results in a dispersionwhere encapsulated aqueous beads comprising the active natamyciningredient are dispersed in the hydrophobic phase. The dispersion iscooled below the melting or dropping point of the hydrophobic phase byany suitable process, which results in the formation of microcapsules.The cooling process can be performed, for example by spray cooling orfluidized bed cooling. The microcapsules comprise a number ofencapsulated aqueous beads, which further contain the natamycin, and theencapsulated aqueous beads are further encapsulated in a solidifiedhydrophobic shell matrix.

An advantage of the present invention is that the natamycin is protectedby the shell and that the release of the natamycin from the capsules canbe controlled. The release rate may be controlled, for instance, by thechoice and the amount of the shell material. Thus, the release rate maybe controlled by the melting of the hydrophobic shell or by thediffusion of water into the capsule and subsequent migration ofnatamycin outside the capsule. The release rate of natamycin from thecapsules may be selected according to the intended use by selecting asuitable encapsulating material. The release of the natamycin from thecapsules of the present invention can be controlled and the release canbe initiated in various ways, for example by heat treatment, e.g. byheating, such as in a microwave oven or baking oven, or by freezing, bystress treatment or by any other suitable process. The release of theactive ingredients from the capsules of the present invention can alsobe sustained or it can happen very slowly.

Based on the present disclosure, the person skilled in the art is ableto select a suitable encapsulation process as well as the right type andamount of shell material to be used in any one specific food applicationbased on the conditions required to protect and to release the natamycinin accordance with the present invention.

The new improved natamycin dosage form of the present invention enablesthe use of natamycin in a wide variety of applications, for example invarious new applications in the food/feed or pharmaceutical fields.

Yet another advantage of the method of the invention is that it enablesa high production capacity to be achieved while the costs are still low.

In the following, the invention will be described in greater detail bymeans of preferred embodiments and with reference to the examples.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a natamycin dosage form comprisingnatamycin which is encapsulated within a physiologically acceptableshell to provide a protected natamycin product. The preferred dosageform comprises natamycin encapsulated in microcapsules.

The main object of the invention is to improve the use of natamycin inthe food industry and, consequently, the shell of the natamycin dosageform of the present invention should be made of a physiologicallyacceptable material suitable for addition to a food product. The shellprovides protection for the natamycin and it should be effective insubstantially retaining said natamycin within said shell duringprocessing of food products. Once introduced into a food product, theshell should be effective in providing slow or delayed release of theencapsulated natamycin into the food product.

Most preferably, the natamycin dosage form of the present invention hasa shell which is effective in protecting the encapsulated natamycin fromdegradation by conditions prevailing in the production of a productwhereto the encapsulated natamycin is added, and/or in protecting foodingredients from unwanted attack by natamycin at the wrong time, as wellas in providing release of natamycin in said finished product.

The term “food” as used in the present specification and claims refersgenerally to edible products and beverages of the food and feedindustry. The edible products in question are mainly nutritive and/orenjoyable products requiring preservation for their storage between thetime of production and eventual use.

The term “physiologically acceptable” likewise refers to materialsacceptable and intended for ingestion in connection with food and feedproducts.

Any suitable food grade coating material may be used as thephysiologically acceptable shell material. However, preferred materialsare selected from the group consisting of hydrophobic materials,hydrocolloid materials and mixtures or combinations thereof.

The preferred hydrophobic material is chosen from resins and lipids andcombinations thereof. The lipids include fatty acids, fats, oils,emulsifiers, fatty alcohols, waxes and mixtures or combinations thereof.The preferred hydrocolloids comprise soluble or dispersible coatingmaterials selected from food grade gums, polysaccharides, proteins,shellac and mixtures or combinations thereof.

Typical hydrocolloid shell materials are selected from cellulosicderivatives (including hydroxy propyl methyl cellulose, carboxy methylcellulose, methyl cellulose, microcrystalline cellulose and mixturesthereof) with or without stearic acid, zein, shellac and mixtures orcombinations thereof.

Generally, the shell on the natamycin dosage form of the invention isprovided by co-processing natamycin with an encapsulating material,which is in an aqueous or lipidic solution or suspension or in a moltenstate.

The natamycin which is to be encapsulated may be in liquid form such asin an aqueous suspension or it may be encapsulated as a dry powder. In apreferred process the shell is provided by co-processing the natamycinwith a molten encapsulating material.

A special form of encapsulated natamycin is provided by a doublyencapsulated natamycin dosage form, which comprises microcapsules havinga solidified hydrophobic shell matrix, encapsulated aqueous beads whichare further encapsulated in the solidified hydrophobic shell matrix, andnatamycin incorporated in the encapsulated aqueous beads.

The percentage of active natamycin in the protected natamycin product ofthe present invention is from 1 to 80% by weight. A preferred amount ofnatamycin is between 15 and 50% and the most preferred amount is between30 and 40% by weight.

The process according to the present invention for producing thenatamycin dosage form of the invention comprises co-processing natamycinwith an encapsulating material to cause said material to encapsulatesaid natamycin within a shell, and recovering a protected natamycinproduct.

The encapsulating process is preferably selected from a fluidized bedprocess, liposome encapsulation, spray drying, spray cooling, extrusion,centrifugal co-extrusion, coacervation and mixtures thereof. Fluidizedbed encapsulation and coacervation are the most preferred processes forproviding the natamycin dosage form of the present invention.

In a preferred fluidized bed encapsulation natamycin is co-processedwith an encapsulating material in an aqueous solution or suspension orin a molten state to provide a shell around the natamycin.

In a preferred coacervation process, an encapsulating materialcomprising a hydrocolloid or a mixture of hydrocolloids is used toprovide the shell.

A special coacervation process of the invention comprises a doubleencapsulation including the steps of providing an aqueous phase andnatamycin incorporated in the aqueous phase, providing a hydrophobicphase in a molten form, incorporating or dissolving an encapsulatingmaterial or mixture of encapsulating materials in the aqueous phase orin the hydrophobic phase, combining the aqueous phase with thehydrophobic phase and homogenizing or mixing the combined phases to forma water-in-oil emulsion, encapsulating the aqueous phase in theemulsion, whereby a dispersion comprising encapsulated aqueous beads isformed and the natamycin is encapsulated in the aqueous beads, andprocessing the dispersion obtained to form microcapsules where theencapsulated aqueous beads are further encapsulated in solidifiedhydrophobic shell material.

The novel natamycin dosage form of the present invention provides anovel method for the preservation of food products, which comprisesadding to a food product an effective food-preserving amount ofnatamycin which is encapsulated within a shell. The encapsulatednatamycin may be added to the food product prior to or in connectionwith the production of the food product. The shell is effective inprotecting the encapsulated natamycin from degradation by conditionsused in the production or storage of the food product and/or inprotecting the ingredients of the food product from the antifungaleffect of the natamycin, and it provides release of natamycin in thefood product.

The encapsulation protects the natamycin from degradation by conditionssuch as heat, light, oxidation and high or low pH.

A special benefit is provided by a preferred embodiment of the inventionwhen the encapsulated natamycin is included in a dough prior to thecooking of a yeast-leavened bakery product since the yeast is protectedby the encapsulating shell from direct contact with the natamycin duringthe leavening.

Furthermore, the encapsulated natamycin is preferably protected againstthe heat of the baking by the shell. Natamycin is degraded by exposureto heat. During baking, which is typically performed at temperaturesranging from 180 to 300° C., natamycin degradation would significantlyreduce the level of active natamycin in the finished baked product. Byselecting an encapsulating material having a sufficient heat stability,the heat degradation of natamycin can be substantially reduced. Duringbaking and/or after baking, the shell releases the natamycin so that thefinished product is effectively protected against fungal attack.

A preferred use of the present invention comprises use of the novelnatamycin dosage form in dough for bread, which is to be sliced forsale. The natamycin released from within the capsule shell in thefinished product protects the individual cut bread slices from fungalattack.

Sliced bread is a very convenient food product for consumers. However,the slicing provides an additional process step in the production, andone which is typically performed after the bread has cooled after bakingwhen the product is very susceptible to fungal attack. When the slicingis performed, contamination may take place and as a result, the slicedproduct will show fungal growth between the slices during storage. Thebread slicing exposes a much greater surface area of the bread tocontamination particularly by molds.

The copending patent application U.S. Ser. No. 10/765,210, filed Jan.28, 2004 and included herein by reference, discloses the protection offine bakery goods by spraying the surface of the goods with natamycinand thus to increase the shelf life of the product. However, it isimpossible to apply natamycin between the slices of sliced bread. Thepresent invention provides a solution to the problem of protectingsliced bread by natamycin.

The present invention provides a preserved food product which comprisesas a preservative an effective food preserving amount of natamycin whichis encapsulated within a shell. Such food products are preferablyselected from salad dressings, dips, salsa sauce, ketchup, purees,pickles, acidic dairy products (including natural cheese, cottagecheese, acidified cheese, cream cheese, yoghurt, sour cream, processedcheese), fruit juices, acidic drinks, alcoholic drinks (including wine),cooked and uncooked bakery products, chilled dough and similar bakeryitems, dairy fillings and toppings for baked goods, surface glazes andcoatings for bakery items and other heat-processed items, marinades,marinated meat or poultry, breaded meat or poultry, fast food products,kits for making snacks or meals, kits for making bakery products, andcombinations thereof. The present invention also provides preserved heatprocessed pet foods and other feed products such as dog or cat food andbroiler feed.

In a preferred embodiment of the invention natamycin is protectedagainst pH attack by a process comprising encapsulating natamycin withina shell material to provide an encapsulated natamycin which is protectedagainst degradation caused by low or high pH, the shell material beingsufficiently resistant to protect the encapsulated natamycin fromdegradation by pH and said shell material providing a slow and/ordelayed release of natamycin.

Natamycin in solution is fairly stable at neutral pH but is easilydegraded, especially at room temperature when the pH rises above pH 10or sinks below pH 4.5, and especially below pH 4. Thus, for instancenatamycin included in acidic products will gradually degrade and willconsequently leave the product unprotected at storage and use. The rateof natamycin degradation increases as the temperature is increased.

Many acidic products, such as salad dressings and condiments are storedat ambient temperature and used during a prolonged space of time evenafter opening of the package. Acidic beverages such as fruit juices canbe stored at ambient temperature and may be open to fungal attack.Marinades and marinated meat and poultry are typically stored for aprolonged time at ambient temperature. Many acidic dairy products arestored at ambient or chilled temperatures and may be spoiled by fungalgrowth. When encapsulated natamycin is added to such products, theencapsulation protects the enclosed natamycin and slowly allows it todiffuse into the product to replace any degraded natamycin thus keepingthe amount of active natamycin at a suitable antifungal level in theproduct.

The encapsulated natamycin of the present invention provides similarbenefits in other acidic products, especially those that are stored atambient temperature.

The encapsulating shell may also be designed to protect the natamycinagainst any heat during processing of the acidic food product, such aspasteurization at temperatures of typically 60 to 120° C. and more often60 to 95° C.

The processes used for the encapsulation are briefly outlined below.

Coacervation is a process which works for both water- and fat-basedapplications since the shell is crosslinked and not soluble in eitherwater or fat.

Fluidized bed coating is suitable for food applications where thecontinuous phase is water, the possible coating materials include lipids(mono-, di-, triglycerides, fatty acids, waxes and mixtures) appliedfrom the melted form, water-insoluble polymers applied from an ethanolicsolution (such as zein and shellac). For applications where thecontinuous phase is fat, the coating materials include natural, modifiedor synthetic hydrocolloids (carrageenan, alginate, pectin, locust beangum (LBG), hydroxypropyl methylcellulose (HPMC), methycellulose) with orwithout additives (such as film forming agents) applied from a watersolution or suspension. The particle size of the natamycin should beover 100 μm, preferably over 150 μm.

A double encapsulation according to the present invention is suitablefor fat-based food applications. The inner phase might be composed ofwater containing a dissolved natamycin/b-cyclodextrin complex and anygelled/crosslinked hydrocolloids or might be composed of glycols (suchas ethylene glycol) containing dissolved natamycin andgelled/crosslinked zein.

In a liposome encapsulation natamycin could be incorporated in thelipidic bilayer of the liposome phase.

Spray cooling is a process suitable for water-based food applications.Natamycin is typically incorporated and suspended in melted lipid(mono-, di-, triglycerides, fatty acids, waxes and mixtures) andatomized in cool air to form solid particles containing encapsulatednatamycin.

Spray drying is suitable for fat-based food applications. Natamycin istypically incorporated and suspended in aqueous solution ofhydrocolloids (gum arabic, modified starch, maltodextrins, wheyproteins, caseinate, or the like) with or without additives (such asemulsifiers) and the mixture is atomized in hot air to evaporate thewater and form a solid particles containing encapsulated natamycin.

Extrusion is a process which is mainly suitable for fat-based foodapplications and centrifugal coextrusion is suitable for water-basedfood applications.

Encapsulation in crosslinked hydrocolloid beads is suitable for bothwater- and fat-based food applications. A suspension of natamycin (aloneor in combination with a suitable solvent) is typically prepared inaqueous alginate, low ester pectin or any other “crosslinkable”hydrocolloids and added dropwise or sprayed into a bath of aqueouscalcium ions. The crosslinked beads or particles containing theencapsulated natamycin are collected by filtration and used as is (wet)or dried by fluidized bed or any other suitable means.

Food products, which are especially suitable for being preserved by thenovel natamycin dosage form of the present invention includefat-containing acidic products such as salad dressings and acidic dairyproducts (natural cheese, cottage cheese, acidified cheese, creamcheese, yoghurt, sour cream). Many of these products can be preservedwith natamycin in non-encapsulated form and they will generally keepwell, if chill stored. However, if they are stored at ambienttemperature, degradation of the natamycin is a problem. This problem issolved by the encapsulated natamycin of the present invention.

In USA salad dressings are usually cold-processed, in which casecontaminant yeasts and moulds are potential spoilage contaminants. Thecombination of ambient temperature storage and low pH causes rapidnatamycin degradation. If non-encapsulated natamycin, which is addedwhen the dressings are first made fails to rapidly kill all thecontaminant yeasts, and if any mould spores are present (these are notnormally killed by natamycin), there is potential for fungalgrowth/spoilage once natamycin levels drop.

By use of the encapsulated natamycin of the present invention the acidicfood products may be stored at ambient temperature for up to 12 months.

The preferred processes for encapsulation for the acidic food productscomprise coacervation and fluidized bed encapsulation.

The coacervation process typically involves 1) preparing a suspension ofnatamycin in an aqueous solution of hydrolloids, 2) decreasing thesolubility of the hydrocolloids, to cause a phase separation and theformation of a hydrocolloid-rich third phase by use of additives or byadjusting the temperature, 3) processing the tri-phasic system in suchas way as to deposit the newly formed coacervate phase onto thesuspended natamycin particles and finally 4) hardening the hydrocolloidshell around the natamycin by adjusting the temperature, adding chemicalor enzymatic crosslinker or otherwise followed by the isolation of themicrocapsules by freeze-drying, spray-drying, filtration or any othermeans.

In the fluidized bed encapsulation the appropriate shell material istypically applied from aqueous solutions or suspensions include HPMC,methylcellulose, microcrystalline cellulose and other cellulosicderivatives with or without stearic acid, other fatty acids, or otherhydrophobic additives. Appropriate shell material applied from themolten state include lipids, mono-, di- or tro-glycerides, fatty acids,fatty alcohols, waxes, or mixture thereof or any other meltablehydrophobic material.

Another type of food product which derives great benefits from thepresent invention is fruit juice and acidic drinks. Benefits are alsoderived for processed fruit, low pH sauces, such as ketchups and purees,salsa sauces, condiments, dips, pickles, etc, alcoholic drinks such aswine or beer and the like. These liquid products may contain fat(acidified fruit milk drinks, etc). They may be pasteurised. Thecombination of pasteurisation at low pH, but more importantly acid pHand ambient temperature storage results in degradation of non-protectednatamycin. If post-processing contamination with yeasts or moulds hasoccurred, or heat-resistant mould spores (e.g. Byssochlamys,Talaryomyces) or yeast ascospores survive the processing, fungal growthwill occur once natamycin levels are degraded.

Animal feed products such as dog and cat food or broiler feed is oftenheat processed during the production thereof and then stored at ambienttemperature. The encapsulated heat-stable natamycin of the presentinvention can conveniently be used to protect such feed products.

In liquid products such as juices or wines, the shell material should bemade of a material which does not disturb the clarity of the liquid.

When the natamycin is added in the form of the novel capsules of thepresent invention, the shell will slowly dose out small amounts ofnatamycin and keep the liquid products free of fungal growth forextended periods (3-9 months) of storage at ambient temperature. Theencapsulation provides a special benefit for heat-treated acidic liquidssince the shell protects the natamycin both from heat and acid attack.

Natamycin is a preservative which may also be used to advantage inbakery products. Most baked goods are susceptible to mould spoilage dueto aerial contamination with mould spores after baking. Propionate iscommonly incorporated into bread and other yeast-leavened doughs as ananti-mould agent. The anti-yeast activity of propionate is much weakercompared to its anti-mould activity in these doughs. Although propionatehas a slight inhibitory effect against the bakers' yeast, this isacceptable.

Natamycin cannot be used in this way because it is strongly activeagainst both yeasts and moulds. Encapsulation of the natamycin preventsthe natamycin activity against the bakers' yeast until after theleavening is complete. It also protects the natamycin during the bakingprocess. This is particularly useful for products such as sliced breadsthat have a large surface area exposed to air contamination.

The encapsulation processes of the present invention are described insome detail below:

1. Fluidized Bed Encapsulation

The natamycin is preferably used in dry powder form. If the rawnatamycin particle size is too fine, the powder can be agglomerated inan suitable equipment using a binder solution (solution of stickyhydrocolloids such as alginate or maltodextrine) in order to obtain adense powder of particle size between 100-500μ. The appropriate powderis then introduced into the coating chamber of a fluidized-bedmicroencapsulation unit and fluidized at inlet air flow rate of 20-135cm/s at the bottom plate and temperature between 5 to 75° C. are used tofluidized the particles. A coating material is then sprayed onto thefluidized bed of antimicrobial using a double fluid nozzle and highpressure atomization air.

In one example, a melted mixture of triglyceride and additives issprayed onto the antimicrobial powder to form a continuous layer of fataround each individual particle as the melted fat spread and solidifieson the particles. The amount of fat applied can be up to 60%, but nousually no lower than 15% w/w.

In another example, a dispersion or solution of coating material inwater and/or ethanol is sprayed onto the fluidized particles and thefluidization air is used to evaporate the solvent or the water, whichleaves behind a continuous film of coating material on the antimicrobialparticles.

Examples of coating material in this case include any hydrocolloids(polysaccharides, proteins, shellac, zein or any other soluble ordispersible coating materials).

2. Liposome Encapsulation.

Typically, liposomes are prepared using a dehydration-hydration methodinvolving organisc solvent, such as the one described below. However,solvent-free methods, more suitable for food ingredients, are alsoavailable using microfluidization or homogenization devices or byrepeated freeze-thaw cycles.

A typical procedure for the preparation of liposome-encapsulatednatamycin involves the preparation of a solution of 1 g of abilayer-forming lipid and 100 mg of cholesterol or alpha-tocopherol in asuitable organic solvent and evaporating the solvent so as to form athin dry lipid film on the bottom of the container.

After thorough drying of the lipid film, 1 L of water containingnatamycin at or over the saturation concentration (natamycin solubilitycan be increased if desired by the formation of alkaline salts) is addedto the container and the mixture is thoroughly mixed or homogenized.

The resulting suspension of multilamellar vesicle (MLV) can be furtherprocessed by microfluidization and or sonication to form smaller morehomogenous small unilamellar vesicle (SUV). The suspension ofliposome-encapsulated natamycin can be added directly to the applicationor dried by lyophilization or any other appropriate drying procedures.

3. Spray Drying

Natamycin can be encapsulated in a matrix of hydrocolloids by means ofspray drying. In a typical procedure, an aqueous natamycin suspension inwhich a hydrocolloid or a mixture of hydrocolloids is dissolved(water-soluble polysaccharides, proteins, modified polysaccharides withor without film forming agents such as oligosaccharides, plasticizers,emulsifiers or other additives) is prepared at near-neutral pH (tominimize degradation of natamycin). Then, the mixture is pumped throughan atomizer (rotary atomizer, pressure nozzle, double fluid nozzle orany other atomization device) into a drying chamber co- orcounter-currently with heated air.

The temperature of the heated air is typically between 160 and 200° C.,can be as high as 300° C., but is preferably in the range of 100-160° C.Evaporation of water yields a free flowing powder of microcapsulescontaining dispersed natamycin in the dry hydrocolloid(s) matrix.

4. Spray Cooling

In spray cooling/chilling/congealing of natamycin, the powderednatamycin is dispersed in a molten lipid or mixture of lipids (mono-,di-, tri-glycerides, esterified glycerides, animal, vegetable or mineralwaxes, any other meltable material at temperature between 45 and 125°C.) with or without the aid of surface-active additives. The dispersionis then pumped through an atomizer (rotary atomizer, pressure nozzle,double fluid nozzle, spinning disk or any other atomization device) intoa cooling chamber co- or counter-currently with cooled air.

The temperature of the cooled air is typically between −10 and 30° C.,but can be as low as −40° C. solidification of the lipid yields a freeflowing powder of microcapsules containing dispersed natamycin in thecrystallized lipidic matrix.

5. Extrusion

Encapsulation of natamycin by extrusion can be achieved by processingthe powdered natamycin (preferably of small particle size) together witha melted or plasticized polymeric shell material in a double- orsingle-screw extruder under pressure, followed by the cooling or thedrying of the mass coming out of the extruder die and milling orcrimpling to the appropriate particle size. The polymeric mass is meltedin the extruder at relatively high temperatures in the presence of smallamount of water, which causes the mass to become flowable. The mass, inwhich the natamycin is incorporated, is extruded and cooling results inthe transformation of the mass into a glassy state which is highlyimpermeable to oxygen and other hydrobobic external agents. Shellmaterials suitable for extrusion of natamycin include oligosaccharides,polysaccharides, modified polysaccharide, proteins or mixtures thereofwith or without the use of plasticizing, emulsifying or stabilizingadditives.

6. Centrifugal Co-Extrusion

Encapsulation of natamycin by centrifugal co-extrusion is a variation ofthe spray cooling process. In centrifugal coextrusion of natamycin, thepowdered antimicrobial is first dispersed in a molten lipid or mixtureof lipids (mono-, di-, tri-glycerides, esterified glycerides, animal,vegetable or mineral waxes, any other meltable material at temperaturebetween 45 and 125° C.) with or without the aid of surface-activeadditives. The dispersion is then pumped through the inner part of adouble fluid nozzle while another stream of molten lipid or mixture oflipids (same as above) is pumped through the outer part of the doublefluid nozzle. The nozzle is rotated around its axis so as to break upthe stream of melted fat in discrete globules, which are solidified bycooled air. The resulting microcapsules are composed of an outer layerof solidified fat encapsulating a core of solidified fat containingdispersed natamycin.

7. Coacervation

The natamycin dosage form of the present invention can be formed bycoacervation. The coacervation of the shell material, such ashydrocolloid, is carried out by using any suitable coacervation process.The coacervation can be performed for example by adding salt(s),sugar(s), or other additives, which cause the phase separation of thehydrocolloid(s). The coacervation can also be performed by subjectingthe emulsion to heating, cooling, pH change by adding acid or base,which cause the phase separation of the hydrocolloid(s). The depositionof the coacervated phase around the aqueous phase is spontaneous anddriven by surface tension forces. The coacervate layer can afterwards besubjected to cross-linking or hardening by any suitable means, which areknown to persons skilled in coacervation.

The shell materials suitable for coacervation are selected form thegroup comprising any mixture of one or many ionic hydrocolloids and oneor many amphoteric hjydrocolloids, such as any mixture ofpolysaccharides and proteins, gelatine/arabic gum, gelatine/CMC, anyproteins/ionic hydrocolloids, any combination of hydrocolloids and asolubility-reducing agent such as salts, sugars, acids or bases.

8. Double Encapsulation

According to a special aspect of the present invention, the natamycinsuspension is double encapsulated in microcapsules. In that case, thenatamycin is first incorporated (suspended) in an aqueous phasecontaining encapsulating material, such as hydrocolloid or any othersuitable encapsulating material or mixture thereof, and the aqueousphase is encapsulated, for example by gelling, cross-linking,coacervation, sintering or by any other suitable means, and theresulting encapsulated aqueous bead or beads is/are further encapsulatedin a solidified hydrophobic shell material.

A hydrophobic shell material is selected based on desired properties ofthe capsules, for example based on the intended use of the capsules,storage temperature, etc. The hydrophobic shell material should have amelting point above 45° C. so that it can be stored at room temperature,in general any hydrophobic material can be used if the capsules arestored below the melting temperature of the hydrophobic material.

In this application, melted form means that the hydrophobic phase is atthe lowest temperature at which the hydrophobic phase is sufficientlyfluid to drip, as determined by test method ASTM D 566 or D 265.

The hydrophobic shell material useful in the various processes of theinvention is selected from the group comprising fats, oils, waxes,resins, emulsifiers or mixtures thereof, which are preferablyfood-grade. Preferably the hydrophobic shell material is selected fromthe group comprising animal oils and fats, fully hydrogenated vegetableor animal oils, partially hydrogenated vegetable or animal oils,unsaturated, hydrogenated or fully hydrogenated fatty acids,unsaturated, partially hydrogenated or fully hydrogenated fatty acidmonoglycerides and diglycerides, unsaturated, partially hydrogenated orfully hydrogenated esterified fatty acids of monoglycerides ordiglycerides, unsaturated, partially hydrogenated or fully hydrogenatedfree fatty acids, other emulsifiers, animal waxes, vegetable waxes,mineral waxes, synthetic waxes, natural and synthetic resins andmixtures thereof.

Animal oils and fats are such as, but not restricted to, beef tallow,mutton tallow, lamb tallow, lard or pork fat, sperm oil. Hydrogenated orpartially hydrogenated vegetable oils are such as, but not restrictedto, canola oil, cottonseed oil, peanut oil, corn oil, olive oil, soybeanoil, sunflower oil, safflower oil, coconut oil, palm oil, linseed oil,tung oil and castor oil. Free fatty acids are such as, but notrestricted to, stearic acid, palmitic acid and oleic acid. Otheremulsifiers are such as, but not restricted to, polyglycerol esters,sorbitan esters of fatty acids. Animal waxes are such as, but notrestricted to, beeswax, lanolin, shell wax or Chinese insect wax.Vegetable waxes are such as, but not restricted to, carnauba,candelilla, bayberry or sugarcane waxes. Mineral waxes are such as, butnot restricted to, paraffin, microcrysalline petroleum, ozocerite,ceresin or montan. Synthetic waxes are such as, but not restricted to,low molecular weight polyolefin, polyol ether-esters and Fisher-Tropschprocess synthetic waxes. Natural resins are such as rosin, balsam,shellac and zein.

The hydrocolloid shell material of the invention is any food-gradehydrocolloid which is susceptible to encapsulation by the processes ofthe invention.

The material is selected from the group comprising hydrocolloids, sodiumalginate, gum arabic, gellan gum, starch, modified starch, guar gum,agar gum, pectin, amidified pectin, carrageenan, xanthan, gelatine,chitosan, mesquite gum, hyaluronic acid, cellulose derivatives such ascellulose acetate phtalate, hydroxy propyl methylcellulose (HPMC),methyl cellulose, ethyl cellulose and carboxy methyl cellulose (CMC),methyl acrylic copolymers, such as Eudragit®, psyllium, tamarind,xanthan, locust bean gum, whey protein, soy protein, sodium caseinate,any food-grade protein, shellac, zein, any synthetic or naturalwater-soluble polymers, and mixtures thereof.

According to a special double encapsulation embodiment of the presentinvention, the microcapsule comprises a solidified hydrophobic shellmatrix, a gelled or cross-linked aqueous hydrocolloid bead or beadsencapsulated in the solidified hydrophobic shell matrix, and natamycinsuspended in the gelled or cross-linked aqueous hydrocolloid bead orbeads.

The gelled hydrocolloids have a gelling temperature above roomtemperature. Examples of gelled hydrocolloids include carrageenan,gelatine, guar gum, agar gum, starch, modified starch and mixture ofxanthan and locust bean gum, mixture of carrageenan and locust bean gumand mixture of any gelling hydrocolloids and other non-gellinghydrocolloids.

The cross-linking of the hydrocolloids is carried out by usingcross-linking agents or by a variety of mechanisms. If the hydrocolloidis a protein or polysaccharide bearing amino groups, it can becross-linked by using dialdehydes, such as glutaraldehyde. If thehydrocolloid is a polysaccharide, such as sodium alginate, gellan gum orpectin, it can be cross-linked with multivalent ions, such as calcium ormagnesium. The cross-linking can also be carried out by othermechanisms, such as heating, pH adjustment, applying pressure or byenzymatic cross-linking. Proteins, for example, can be cross-linked bysubjecting a protein to a high pressure, preferably from 5 to 200 bar,and/or by subjecting a protein to a temperature which is above thedenaturation temperature of the protein. The enzymatic cross-linking ofproteins can be carried out for example with transglutamidase. Based onthe hydrocolloid used, a person skilled in the art is able to decidewhich method of gelling or cross-linking is used.

The capsules of the present invention are preferably microcapsules andcomprise 1 to 80%, preferably 15 to 50%, most preferably 30 to 40%natamycin encapsulated in the hydrophobic or hydrocolloid shell. Thesize of a microcapsule is approximately between 40 to 800 microns,preferably 100 to 150 microns. The amount of natamycin encapsulatedwithin the shell of a microcapsule may vary, depending on the intendeduse of the microcapsules. The size of the microcapsules of the presentinvention may also vary depending on the intended use.

The aqueous phase mentioned in the present specification means water ora mixture of water and any other water-miscible solvents, such asethanol, ethylene glycol or glycerol. The aqueous phase may also containadditives, such as carbohydrates, such as monosaccharides oroligosaccharides to modify the properties of the hydrocolloid gel,inorganic salts to modify the properties of protein gels, preservativesto avoid deterioration of the microcapsules by bacteria or fungus oremulsifiers as processing aids, sorbitan tristearate or otheremulsifiers as crystal form modifier, hydrophobic natural or syntheticpolymers to modify mechanical properties of the capsule, plastizisers,preservatives to avoid deterioration of the capsules.

The encapsulated natamycin described above can be used in a wide varietyof applications in food industry and in pharmaceutical applications.

The capsules of the present invention can be used in a great variety ofapplications, depending for example on the properties of the capsules,the hydrocolloid, the hydrophobic material or the size of the capsules.A controlled release of the active ingredients from the capsules can beachieved by the present invention. The release of the active ingredientsfrom the capsules can be controlled by initiating the release in variousways, for example by heat treatment, by heating in a microwave oven orbaking oven, by pH, by light, or by any other suitable process. Therelease of the active ingredients from the capsules of the presentinvention can also happen very slowly. The release of the natamycin mayalso take place upon freezing of the capsules. Freezing causes any waterphase inside the capsule to expand, which causes the external shellmaterial to crack. Upon thawing, the natamycin is quickly released fromthe microcapsule.

In bakery, for example, delayed release of natamycin can be achievedwith the capsules of the present invention. This is very important inorder to avoid inhibition of the required activity of the baker's yeast.Increased heat stability of the natamycin is achieved for example inpasteurised or heat-processed foods. Delayed release of natamycin isalso very important for other yeast fermented foods.

The present invention relates to the use of encapsulated natamycin aspreservative agent providing slow, controlled and/or sustained releaseof the natamycin.

Controlled release of natamycin in food products, such as baked goods,pizza, is achieved with the capsules of the present invention. Theencapsulated natamycin is retained in the product until heat, pH, lightand/or stress treatment is applied to release the natamycin. Heat can beprovided for example by a micro-wave-oven, conventional oven or hotwater. Stress can be provided for example by processing conditions ormastication.

Slow release of natamycin for example in processed meat products or inbeverages, such as orange juice, is achieved with the encapsulatednatamycin of the present invention. The natamycin preservative agent inthe capsules of the present invention is slowly released in the productas it is naturally degraded. This effectively prevents growth of fungior other undesirable micro organisms for a longer period of time thannon-encapsulated natamycin, thus ensuring a longer shelf life for thefood product. The shell can also provide thermal stability to natamycinso as to survive heat treatment and harsh processing conditions, but toremain active during storage of the processed product.

Delayed release of natamycin in bakery applications is achieved by thecapsules of the present invention. Natamycin is useful for extending theshelf life of breads and other bakery products, but at the expense ofdetrimentally affecting the effectiveness of the yeast. The delayedrelease allows a more efficient use of the yeast, while also providingthe preservative properties after the natamycin is released duringbaking.

The encapsulated natamycin is also useful in many ready-to-use foodproducts, in snacks and in kits for producing snacks and meals. Theencapsulated natamycin ensures the slow and continuous release ofnatamycin into the product so as to keep the level of active(non-degraded) natamycin high enough to prevent spoilage of the product.

The following examples serve to illustrate the invention

EXAMPLES Example 1

Production of Encapsulated Natamycin by a Coacervation Process

First, a solution of gelatine (219 g, isoelectric point=8) in 6 litersof water at 50° C. was prepared. Secondly, a solution of 219 g of gumacacia was dissolved in 6 L of water at 50° C. The two solutions weremixed together and kept at 45° C. under vigorous stirring. 700 g ofNatamax™ SF (Danisco) was added to the aqueous solutions and the pH wasrapidly lowered to 4.0 using 1M HCl, after which the temperature waslowered to 5° C. at the rate of approximately 1° C./min, maintaining thestirring throughout. 36 ml of an 1:1 aqueous solution of glutaraldehydewas added, the pH was re-adjusted to 8.5 using aqueous 1M NaOH and thenthe temperature was increased back to 45° C. at a rate of approx 2°C./min. Finally, the whole mixture was spray dried in a spray towerusing a double-fluid nozzle mounted in the fountain configuration, airinlet temperature of 180° C. and a spray rate to maintain the outlet airtemperature of about 100° C.

In an alternatively process, 1 kg each of gum arabic and maltodextrin(DE 12) are dissolved in the aqueous mixture just prior to spray drying.

Example 2

Fluid Bed Encapsulation of Natamycin Preprocessing.

If the natamycin particle size is too fine (below 100 micrometersaverage), the powder is agglomerated to a larger average particle sizefor easier processing by fluidized bed. Larger average particle size notonly makes the process easier, but also allow the use of less coatingmaterial while achieving the same protection as with more shellmaterial. Natamycin is agglomerated in an suitable equipment such as ahigh shear mixer (such as a Lödige mixer using a binder solution(solution of sticky hydrocolloids such as alginate or maltodextrine) inorder to obtain a dense powder of particle size above 150, preferablybetween 200-350 μm and bulk density above 0.4, preferably above 0.7g/cm³.

Hotmelt Fluid Bed Encapsulation

3 kg of agglomerated natamycin is introduced into the coating chamber ofa Aeromatic-Fielder MP1 fluidized-bed microencapsulation unit andfluidized using inlet air flow rate of 80 cm/s and temperature of 43° C.A melted hydrogenated triglyceride kept at 85° C. is then sprayed ontothe fluidized bed of antimicrobial using a peristaltic pump and a doublefluid nozzle set a 2 bar and 2 m³ of air/h. The fat is applied at around1 kg/h, in such a way to form a continuous layer of fat around eachindividual particles as the melted fat spread and solidifies on theparticles. Enough fat is applied to reach a final product containing 30%fat and 70% natamycin.

Example 3

Extrusion Encapsulation of Natamycin

A mixture of 60 parts of corn starch, 25 parts of natamycin and 10 partsof polyethyleneglycol and 5 parts of water is mixed together andintroduced in a clextral double-screw extruder, the first barrel heatedto 40° C. The mass is treated at 100° C. for just a few seconds inbarrels 2 and 3 then cooled down to 45° C. in barrels up to the die.Alternatively, a vacuum pump is installed on the last barrel so as toget rid of the water. The exiting rope is cut into pieces between 250and 500 μm.

Example 4

Use of Encapsulated Natamycin in Orange Juice

Natamycin was encapsulated by a coacervation method as described inExample 1, using either gelatine and acacia as a shell material(NAP03015), or gelatine, acacia and maltodextrin (NAP03023).

The samples, together with natamycin as Natamax™ (Danisco) were added toorange juice (pH 3.85) and heated at 100° C. for 10 minutes. Theresidual natamycin levels in the juice before and after treatment weretested by HPLC. Samples were diluted in methanol for this assay.

The results are shown in Table 1.

The experiment shows that the microcapsule prevented release ofnatamycin, so that not all the estimated natamycin present could bedetected before the heating step. After heating, the encapsulatednatamycin showed recovery levels higher than with the unprotectednatamycin. TABLE 1 Heat protection of encapsulated natamycin in orangejuice Theoretical Detectable Detectable payload Actual natamycin innatamycin in based on natamycin juice before juice after pure addedheating/ppm heating/ppm natamycin Addition (based on estimated (% ofnatamycin (% of natamycin Sample W/w level payload) added) added)Natamax ™ 50%  40 ppm 20 ppm 19.7 (98.5%)  5.2 (26%) NAP03015 80%  80ppm 64 ppm 33.0 (52%) 21.1 (33%) NAP03023 36% 140 ppm 50 ppm 14.1 (28%)14.8 (30%)

Example 5

Use of Encapsulated Natamycin in Vinaigrette

A vinaigrette dressing was prepared containing water (494.6 ml), 10%vinegar (220 ml), sugar (90 g) and salt (10 g), pH 2.6. Additions ofencapsulated and unencapsulated natamycin were made as shown in Table 2.Sample NAP03015 was encapsulated by coacervation as described inExample 1. Sample NAP03007 was encapsulated by spray-cooling with ashell material of triglyceride. TABLE 2 Theoretical Actual natamycinpayload of pure Addition added (based on Sample natamycin levelestimated payload) Natamax ™ 50% 40 ppm 20 ppm NAP03007 40% 100 ppm  40ppm NAP03015 80% 50 ppm 40 ppm

The vinaigrette was incubated at 25° C., and samples assayed forresidual natamycin content at regular intervals. The vinaigrette wasshaken before each sampling, and a sample taken for HPLC analysis, whichwas diluted 1:1 in methanol. The natamycin levels found in the mixedvinaigrette and in the water layer only are shown in Table 3 and 4. Theresults show that encapsulation protects the natamycin from aciddegradation in the vinaigrette, allowing a slow release of thepreservative with time. Sample NAP03007 contained only a small amount ofunencapsulated natamycin at the beginning of the experiment. TABLE 3Detectable natamycin in a vinaigrette dressing at 25° C. (Sample takenfrom homogenised dressing) Natamycin percentage of estimated additionlevel (based on estimated addition level) Days at 25° C. NatamaxNAPO3007 NAPO3015 0 70.5%  1.8%   70% 1   38%  4.5% 50.5% 6 22.5% 19.3%23.8% 9   13% 29.5% 36.5% 14   10% 40.8%   29% 21  4.5% 17.5% 10.2%

TABLE 4 Detectable natamycin from the water phase of a vinaigrettedressing at 25° C. Natamycin % of estimated addition level. (based onestimated addition level) Days at 25° C. Natamax NAPO3007 NAPO3015 0 48%  1.5% 13% 1  25% 2.25% 15.3% 6   8% 2.75%  7.8% 9   8%   13%   5%14   6% 13.5%  5.3% 21 2.5% 11.8%  4.3%

Example 6

Use of Encapsulated Natamycin in Bread

A bread is made by preparing a dough containing flour, water, yeast,salt and a dough conditioner. Included in the dough mix is eithernatamycin or encapsulated natamycin or neither. Both natamycinpreparations are added at a potency dosage of 12 ppm (0.0012%) on flourweight and these are added together with the other dry ingredients. Allingredients are mixed together thoroughly for between 3 and 10 minutes.

The dough is then given a short resting period after mixing (approx. 5to 10 minutes) followed by scaling at the required weight. A second restperiod is then applied following a second moulding in shape the dough asdesired. The dough is then placed into a tin or tray. A leavening periodfor about 50 minutes at 85% relative humidity at 40° C. then follows.

The fully proved dough is then baked at between 190 and 230° C. forapproximately 15 to 30 minutes.

Bread containing unencapsulated natamycin shows poor leavening, whereasleavening of the encapsulated natamycin proceeds in a similar fashion tothe control bread not containing any natamycin. This demonstrates thebenefit of encapsulation, which prevents the natamycin from inhibitingthe yeast fermentation reaction.

When the bread is cool, the natamycin content in the bread is assayed.The natamycin content from bread containing encapsulated natamycin ishigher than that in the bread containing unencapsulated natamycin,indicating the heat protective benefit of encapsulated natamycin. Thebread is then sliced and observed over the normal shelf life period forgrowth of moulds. Delay of mould spoilage is observed for breadcontaining natamycin. This extension of shelf life is greater for breadcontaining encapsulated natamycin, which is a reflection of the highernatamycin levels surviving the baking process.

Example 7

Encapsulation of Natamycin in a Double Shell

First, a solution of 15 g kappa-carrageenan in 1000 ml of phosphatebuffer at pH 7.0 is prepared at 85° C. To this is added 300 g ofcommercial natamycin (Natamax™ SF, Danisco). The resulting mixture isthoroughly mixed. At the same time, a mixture of 1333 g of a vegetabletriglyceride (GRINDSTED® PS 101, m.p. 58° C.) and 73 g of acetylatedemulsifier (Acetem 50 00) is melted at 85° C. in a water bath. Themelted fat mixture is kept under homogenization (Silverson mixer, 8000rpm) as the aqueous mixture is slowly incorporated. The homogenizationis maintained for 5 minutes after the whole aqueous mixture is added andthen a solution of 3 g of polysorbate 80 in 40 ml of water is addedunder constant mixing. The resulting low-viscosity water-in-oil emulsionis then immediately spray cooled in a Niro spray tower using thefollowing parameters: inlet air temperature 10° C., outlet airtemperature 28° C., rotating atomization wheel speed 10 000 rpm. A freeflowing powder is obtained. The incorporation of encapsulated natamycinin an orange juice results in a much more stable natamycin formulationcompared to when unencapsulated natamycin is used in the liquid, thusdramatically improving survival rate of the natamycinin the beverage.The encapsulated natamycin, as presented in this example, is released ata rate of only 7% after three days.

It will be obvious to a person skilled in the art that as technologyadvances, the inventive concept can be implemented in various ways. Theinvention and its embodiments are not limited to the examples describedabove but may vary within the scope of the claims.

1. A natamycin dosage form comprising microcapsules where natamycin is encapsulated within a physiologically acceptable shell to provide a protected food preservative natamycin product.
 2. A natamycin dosage form according to claim 1, wherein said shell is effective in substantially retaining said natamycin within said shell during processing of said food product.
 3. A natamycin dosage form according to claim 1, wherein said shell is effective in providing slow or delayed release of said encapsulated natamycin into said food product.
 4. A natamycin dosage form according to claim 1, wherein said shell is effective in protecting said encapsulated natamycin from degradation by conditions prevailing in the production of a product whereto said encapsulated natamycin is added and in providing release of natamycin in said finished product.
 5. A natamycin dosage form according to claim 1, wherein said encapsulation is provided by a process selected from a fluidized bed process, liposome encapsulation, spray drying, spray cooling, extrusion, co-extrusion, coacervation and combinations thereof.
 6. A natamycin dosage form according to claim 1 wherein said shell is made of a material selected from the group consisting of hydrophobic materials, hydrocolloid materials and mixtures or combinations thereof.
 7. A natamycin dosage form according to claim 6 wherein said hydrophobic material is chosen from lipids and resins including fatty acids, fats, oils, emulsifiers, fatty alcohols, waxes and mixtures or combinations thereof.
 8. A natamycin dosage form according to claim 7, wherein said hydrophobic material is selected from the group consisting of food grade animal oils and fats, fully hydrogenated vegetable or animal oils, partially hydrogenated vegetable or animal oils, unsaturated, hydrogenated or fully hydrogenated fatty acids, unsaturated, partially hydrogenated or fully hydrogenated fatty acid monoglycerides and diglycerides, unsaturated, partially hydrogenated or fully hydrogenated esterified fatty acids of monoglycerides or diglycerides, unsaturated, partially hydrogenated or fully hydrogenated free fatty acids, other emulsifiers, animal waxes, vegetable waxes, mineral waxes, synthetic waxes, natural and synthetic resins and mixtures thereof.
 9. A natamycin dosage form according to claim 6 wherein said hydrocolloid comprises a soluble or dispersible coating material selected from food grade gums, polysaccharides, proteins, shellac and mixtures or combinations thereof.
 10. A natamycin dosage form according to claim 9, wherein said hydrocolloid is selected from cellulosic derivatives including hydroxy propyl methyl cellulose, cellulose acetate phthalate, carboxy methyl cellulose, methyl cellulose and microcrystalline cellulose, sodium alginate, gum arabic, gellan gum, guar gum, agar gum, pectin, amidified pectin, carrageenan, gelatine, chitosan, mesquite gum, hyaluronic acid, methyl acrylic copolymers, such as Eudragit®, psyllium, tamarind, xanthan, locust bean gum, wellan gum, zein, shellac, whey protein, soy protein, sodium caseinate, synthetic or natural water-soluble polysaccharides, proteins and other hydrocolloids, with or without fatty acids, fatty alcohol, plasticizers including glycerol, polyethyleneglycol and other low molecular weight hydrophilic alcohols, or combinations of any of said hydrocolloids.
 11. A natamycin dosage form according to claim 1 wherein said shell is provided by co-processing natamycin with an encapsulating material, which is in an aqueous or lipidic solution or suspension or in a molten state.
 12. A natamycin dosage form according to claim 11, wherein said natamycin is in aqueous suspension or comprises a dry powder.
 13. A natamycin dosage form according to claim 1, which comprises microcapsules having a solidified hydrophobic shell matrix, encapsulated aqueous beads which are further encapsulated in the solidified hydrophobic shell matrix, and natamycin incorporated in the encapsulated aqueous beads.
 14. A natamycin dosage form according to claim 1, wherein the percentage of active natamycin in said protected natamycin product is from 1 to 80% by weight.
 15. A natamycin dosage form according to claim 14, wherein said percentage is between 15 and 50% by weight.
 16. A natamycin dosage form according to claim 15, wherein said percentage is between 30 and 40% by weight.
 17. A process for preparing a natamycin dosage form comprising co-processing natamycin with a physiologically acceptable encapsulating material to cause said material to encapsulate said natamycin within a shell, and recovering a protected food preservative natamycin product.
 18. A process according to claim 17, wherein said encapsulation process is selected from a fluidized bed process, liposome encapsulation, spray drying, spray cooling, extrusion, co-extrusion, coacervation and mixtures thereof.
 19. A process according to claim 17 wherein said encapsulating material comprises a material selected from the group consisting of hydrophobic materials, hydrocolloid materials and mixtures or combinations thereof.
 20. A process according to claim 17, wherein said encapsulation process comprises fluidized bed encapsulation of natamycin with an encapsulating material in an aqueous solution or suspension or in a molten state.
 21. A process according to claim 17, wherein said encapsulation process comprises coacervation of natamycin with an encapsulating material.
 22. A process according to claim 19, wherein said encapsulating material comprises a hydrocolloid or a mixture of hydrocolloids.
 23. A process according to claim 17, which includes the steps of a) providing an aqueous phase and natamycin incorporated in the aqueous phase, b) providing a hydrophobic phase in a molten form, c) incorporating or dissolving an encapsulating material or mixture of encapsulating materials in the aqueous phase or in the hydrophobic phase d) combining the aqueous phase with the hydrophobic phase and homogenizing or mixing the combined phases to form a water-in-oil emulsion, e) encapsulating the aqueous phase in the emulsion, whereby a dispersion comprising encapsulated aqueous beads is formed and the natamycin is encapsulated in the aqueous beads, and f) processing the dispersion obtained in step e) to form microcapsules where the encapsulated aqueous beads are further encapsulated in solidified hydrophobic shell material.
 24. A method for the preservation of a food product comprising adding to said food product an effective food-preserving amount of natamycin which is encapsulated within a physiologically acceptable shell.
 25. A method according to claim 24, wherein said encapsulated natamycin is added to said food product prior to or in connection with the production of said food product and said shell is effective in protecting said encapsulated natamycin from degradation by conditions used in the production or storage of said food product said shell providing release of natamycin in said food product.
 26. A method according to claim 25, wherein said conditions are selected from natamycin-degrading heat and high or low pH.
 27. A method according to claim 24 wherein said food product is selected from a salad dressing, a condiment, a ketchup, puree, a salsa sauce, a pickle, a dip, an acidic dairy product including natural cheese, cottage cheese, acidified cheese, cream cheese, yoghurt, sour cream and processed cheese, a fruit juice, an acidic drink, an alcoholic drink including wine and beer, a chilled dough, a cooked or uncooked bakery product, a dairy filling or topping, a surface glaze or coating a marinade, marinated or breaded meat or poultry, a pizza topping or base, a fast food product, a kit for making a snack or a meal, a kit for making a bakery product, combinations thereof, pet food and broiler feed.
 28. A method according to claim 24 wherein said encapsulated natamycin is included in a dough for a yeast-leavened or non-yeast-leavened bakery product.
 29. A method according to claim 28, wherein said dough is baked into bread and subsequently sliced.
 30. A preserved food product which comprises as a preservative an effective food preserving amount of natamycin which is encapsulated within a physiologically acceptable shell.
 31. A food product according to claim 30 wherein said food product is selected from a salad dressing, a condiment, a ketchup, a puree, a salsa sauce, a pickle, a dip, an acidic dairy product including natural cheese, cottage cheese, acidified cheese, cream cheese, yoghurt, sour cream and processed cheese, a fruit juice, an acidic drink, an alcoholic drink, a chilled dough, a cooked or uncooked bakery product, a dairy filling or topping, a surface glaze or coating, a marinade, marinated meat or poultry, breaded meat or poultry, a pizza topping or base, a fast food product, a kit for making a snack or meal, a kit for making a bakery product, combinations thereof, pet food and broiler feed.
 32. A food product according to claim 31 wherein said bakery product is sliced or cut bread. 